Ang-2 Expression Research Articles

Angiopoietin-1 attenuates lipopolysaccharide-induced endotoxemia in a Hirschsprung's disease murine model by improving intestinal vascular integrity: implications for treating postoperative Hirschsprung-associated enterocolitis.

Angiopoietin-1 (Ang1) mitigates inflammation as a proangiogenic growth factor. Action of Ang1 on lipopolysaccharide (LPS)-induced endotoxemic inflammation was investigated in endothelin receptor-B null Hirschsprung's disease mice (KO). LPS or saline was injected intraperitoneally in KO (KO-LPS; n = 9, KO-sal; n = 5) and wild-type (WT) (WT-LPS; n = 6, WT-sal; n = 6) pups obtained within 24h of birth. Normoganglionic terminal ileum harvested 6h after LPS was used for RNA extraction and histology. IL-1β, SELE, VEGFA, Ang1, Angiopoietin-2 (Ang2), and TIE2 expression analyzed by quantitative polymerase chain reaction (qPCR), vascular permeability assessed by the Miles assay, severity of inflammation, and immunofluorescence for phospho-TIE2 and VE-cadherin were used to assess endothelial cell contact integrity and compared with KO pups pretreated with intraperitoneal Ang1 [Ang1(KO-LPS); n = 5] or saline [sal(KO-LPS); n = 6] 2h before LPS. KO-LPS pups showed significantly increased inflammation (p < 0.05) and expression of IL-1β, SELE, VEGFA, and Ang2 (p = 0.019, 0.003, 0.008 and < 0.0001, respectively); expression of Ang1 and TIE2 remained unchanged when compared with KO-saline. In Ang1(KO-LPS) ileum, changes seen in sal(KO-LPS) were eliminated and phospho-TIE2 and VE-cadherin fluorescence increased. Ang1 successfully attenuated LPS-induced normoganglionic intestinal inflammation, downregulated pro-inflammatory genes, and improved vascular barrier integrity in KO pups.

Angiopoietin-2 reverses endothelial cell dysfunction in progeria vasculature.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging disorder in children caused by a point mutation in the lamin A gene, resulting in a toxic form of lamin A called progerin. Accelerated atherosclerosis leading to heart attack and stroke are the major causes of death in these patients. Endothelial cell (EC) dysfunction contributes to the pathogenesis of HGPS related cardiovascular diseases (CVD). Endothelial cell-cell communications are important in the development of the vasculature, and their disruptions contribute to cardiovascular pathology. However, it is unclear how progerin interferes with such communications that lead to vascular dysfunction. An antibody array screening of healthy and HGPS patient EC secretomes identified Angiopoietin-2 (Ang2) as a down-regulated signaling molecule in HGPS ECs. A similar down-regulation of Ang2 mRNA and protein was detected in the aortas from an HGPS mouse model. Addition of Ang2 to HGPS ECs rescues vasculogenesis, normalizes endothelial cell migration and gene expression, and restores nitric oxide bioavailability through eNOS activation. Furthermore, Ang2 addition reverses unfavorable paracrine effects of HGPS ECs on vascular smooth muscle cells. Lastly, by utilizing adenine base editor (ABE)-corrected HGPS ECs and progerin-expressing HUVECs, we demonstrated a negative correlation between progerin and Ang2 expression. Lastly, our results indicated that Ang2 exerts its beneficial effect in ECs through Tie2 receptor binding, activating an Akt-mediated pathway. Together, these results provide molecular insights into EC dysfunction in HGPS and suggest that Ang2 treatment has potential therapeutic effects in HGPS-related CVD.

USP9X-enriched MSC-sEV inhibits LSEC angiogenesis in MASH mice by downregulating the IκBα/NF-κB/Ang-2 pathway

Pathological angiogenesis of liver sinusoidal endothelial cells (LSEC) plays a crucial role in the progression of metabolic dysfunction-associated steatohepatitis (MASH)-induced liver fibrosis. Mesenchymal stem cell-derived small extracellular vesicles (MSC-sEV) have shown promising therapeutic potential against MASH. This study aimed to investigate the impact of MSC-sEV on LSEC angiogenesis and elucidate the underlying molecular mechanisms. The effects of MSC-sEV on LSEC angiogenesis were evaluated in Tumor Necrosis Factor- alpha (TNF-α)-treated LSECs in vitro and in Methionine and Choline Deficient Diet (MCD)-induced MASH mice in vivo. Herein, we found that MSC-sEV effectively suppressed LSEC angiogenesis by targeting the angiogenesis marker Angiogenin 2 (Ang-2) in both TNF-α-treated LSECs and MASH mice. Gene manipulation experiments revealed that the primary mechanism by which MSC-sEV inhibited LSEC angiogenesis was through the modulation of nuclear factor kappa B inhibitor alpha (IκBα) / nuclear factor kappa B (NF-κB) / Ang-2 pathway. Additionally, mass spectrometry and co-immunoprecipitation (Co-IP) data suggested that MSC-sEV delivered the ubiquitin specific peptidase 9 X-linked (USP9X) protein to LSECs, leading to enhanced IκBα deubiquitination and NF-κB in activation, ultimately resulting in the inhibition of Ang-2-mediated LSEC angiogenesis. Knockdown of USP9X attenuated the regulatory effects of MSC-sEV on Ang-2 expression, LSEC angiogenesis, and the progression of MASH. In conclusion, our findings indicate that USP9X delivered via MSC-sEV can suppress LSEC angiogenesis and alleviate MASH-induced liver fibrosis through the IκBα/NF-κB/Ang-2 signaling pathway.

Single-cell and spatial transcriptomics reveal apelin/APJ pathway's role in microvessel formation and tumour progression in hepatocellular carcinoma.

The apelin receptor (APJ) is a key player in tumour angiogenesis, but its role in hepatocellular carcinoma (HCC) remains unclear. This study aims to elucidate the function of the apelin/APJ pathway in HCC using a multi-omics approach and identify potential therapeutic biomarkers. Differentially expressed genes related to the apelin/APJ axis were identified from bulk transcriptomics to reveal HCC-associated disparities. Single-cell and spatial transcriptomics were used to localize and analyse the function of these genes. Machine learning models were constructed to predict outcomes based on apelin/APJ expression, and experimental validation was conducted to explore the pathway's impact on HCC angiogenesis. Single cell analysis revealed an overexpression of APJ/Aplin in the endothelium. The stemness of endothelial cell (EC) with high apelin/APJ was enhanced, as well as the expression of TGFb, oxidative stresses and PI3K/AKT pathway genes. Spatial transcriptomics confirmed that EC populations with high APJ scores were enriched within the tumour. Machine learning models showed high prognostic accuracy. High APJ expression was linked to worse outcomes (p = 0.001), and AUC values were high (1 year, 3 year, 5 year) (0.95, 0.97, 0.98). Immune suppression and non-responsiveness of immune therapy were also seen in high-risk groups. The experimental validation showed that silencing apelin reduced angiogenesis (p < 0.05), endothelial proliferation, decreased expression of ANG2, KLF2, VEGFA and lower ERK1/2 phosphorylation. Apelin may serve as a potential therapeutic target in HCC, given its role in promoting tumour angiogenesis and poor patient outcomes.

New Anti-Angiogenic Therapy for Glioblastoma With the Anti-Depressant Sertraline.

Anti-angiogenic therapies prolong patient survival in some malignancies but not glioblastoma. We focused on the relationship between the differentiation of glioma stem like cells (GSCs) into tumor derived endothelial cells (TDECs) and, anti-angiogenic therapy resistance. Especially we aimed to elucidate the mechanisms of drug resistance of TDECs to anti-angiogenic inhibitors and identify novel anti-angiogenic drugs with clinical applications. The mouse GSCs, 005, were differentiated into TDECs under hypoxic conditions, and TDECs had endothelial cell characteristics independent of the vascular endothelial growth factor (VEGF) pathway. Invivo, inhibition of the VEGF pathway had no anti-tumor effect and increased the percentage of TDECs in the 005 mouse model. Novel anti-angiogenic drugs for glioblastoma were evaluated using a tube formation assay and a drug repositioning strategy with existing blood-brain barrier permeable drugs. Drug screening revealed that the antidepressant sertraline inhibited tube formation of TDECs. Sertraline was administered to differentiated TDECs invitro and 005 mouse models invivo to evaluate genetic changes by RNA-Seq and tumor regression effects by immunohistochemistry and MRI. Sertraline reduced Lama4 and Ang2 expressions of TDEC, which play an important role in non-VEGF-mediated angiogenesis in tumors. The combination of a VEGF receptor inhibitor axitinib, and sertraline improved survival and reduced tumor growth in the 005 mouse model. Collectively, our findings showed the diversity of tumor vascular endothelial cells across VEGF and non-VEGF pathways led to anti-angiogenic resistance. The combination of axitinib and sertraline can represent an effective anti-angiogenic therapy for glioblastoma with safe, low cost, and fast availability.

Ang-1 promotes tumorigenesis and mediates the anti-cancer effects of Artesunate on Choroidal melanoma via the regulation of Akt/mTOR signaling pathway

The impact of Ang-1 on tumors remains a subject of contention, with its mechanism of action exhibiting complexity in the progression of diverse tumor types. Ang-1 has been shown to promote the progression of glioma, glioma, esophageal and human cervical cancer, whereas it exerts inhibitory effects on the growth of breast and colon cancer. However, the specific function of Ang-1 in CM has not been clarified. This research aims to explore the function of Ang-1 on CM and the underlying mechanism. WB and qPCR were utilized to measure the expression levels of different factors in CM cells. Clonogenic, CCK-8 and Transwell migration assay were used to probe CM cells’ proliferation and migration ability. Xenograft tumor model was used to testify the effect of Ang-1 and Artesunate (ART) on the growth of CM in vivo. We found Ang-1 promoted CM proliferation and migration, while it was inhibited by ART in vitro. Moreover, both ART treatment and Ang-1 knockdown had the effect of suppressing tumor growth in CM xenograft model. Mechanically, Ang-1 activated Akt/mTOR pathway and induced epithelial-mesenchymal transition (EMT) in CM cells. Furthermore, ART regulated Akt/mTOR pathway by decreasing the expression of Ang-1 in CM cells. Ang-1 promotes tumorigenesis of CM by regulating Akt/mTOR pathway, which can be inhibited by ART.

Analysis of Expression of the ANG1, CaSR and FAK Proteins in Uterine Fibroids.

Understanding the molecular factors involved in the development of uterine myomas may result in the use of pharmacological drugs instead of aggressive surgical treatment. ANG1, CaSR, and FAK were examined in myoma and peripheral tissue samples taken from women after myoma surgery and in normal uterine muscle tissue samples taken from the control group. Tests were performed using tissue microarray immunohistochemistry. No statistically significant differences in ANG1 expression between the tissue of the myoma, the periphery, and the normal uterine muscle tissue of the control group were recorded. The CaSR value was reduced in the myoma and peripheral tissue and normal in the group of women without myomas. FAK expression was also lower in the myoma and periphery compared to the healthy uterine myometrium. Calcium supplementation could have an effect on stopping the growth of myomas.

Differential Impact of Tumor Endothelial Angiopoietin-2 and Podoplanin in Lymphatic Endothelial Cells on HCC Outcomes with Tyrosine Kinase Inhibitor Treatment According to Sex.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide. Curative treatments are available to a minority of patients, as HCC is often diagnosed at an advanced stage. For patients with unresectable and multifocal HCC, tyrosine kinase inhibitor drugs (TKIs) are the only potential treatment option. Despite extensive research, predictors of response to these therapies remain elusive. This study aimed to analyze the biological and histopathological characteristics of HCC patients treated with TKIs, focusing on angiogenesis and lymphangiogenesis. Immunohistochemistry quantified the expression of angiopoietin-2 (Ang2), lymphatic endothelial cells (LEC) podoplanin, and C-type Lectin Domain Family 2 (CLEC-2), key factors in neoangiogenesis and lymphangiogenesis. An increased expression of endothelial Ang2 and LEC podoplanin predicted a lower risk of metastasis. Female patients had significantly longer overall survival and survival on TKIs, associated with higher tumor expression of endothelial Ang2 and LEC podoplanin. Moreover, LEC podoplanin expression and a longer time on TKIs were independently correlated with improved survival on TKI therapy at Cox regression analysis. These findings suggest that endothelial Ang2 and LEC podoplanin could be potential biomarkers for predicting treatment outcomes and guiding therapeutic strategies in HCC patients treated with TKIs.

Promotion of mature angiogenesis in ischemic stroke by Taohong Siwu decoction through glycolysis activation.

Backgrounds: Mature angiogenesis plays a critical role in improving cerebral ischemia-reperfusion injury (CIRI). Glycolysis serves as the primary energy source for brain microvascular endothelial cells (BMECs), whereas other vascular cells rely on aerobic respiration. Therefore, intercellular variations in energy metabolism could influence mature angiogenesis. Taohong Siwu Decoction (THSWD) has demonstrated efficacy in treating ischemic stroke (IS), yet its potential to promote mature angiogenesis through glycolysis activation remains unclear. Methods: In this study, we established a middle cerebral artery occlusion/reperfusion (MCAO/R) model in vivo and an oxygen-glucose deprivation/reoxygenation (OGD/R) model in vitro. We assessed neuroprotective effects using neurobehavioral scoring, 2,3,5-triphenyltetrazolium chloride (TTC) staining, Hematoxylin-eosin (HE) staining, and Nissl staining in MCAO/R rats. Additionally, we evaluated mature angiogenesis and glycolysis levels through immunofluorescence, immunohistochemistry, and glycolysis assays. Finally, we investigated THSWD's mechanism in linking glycolysis to mature angiogenesis in OGD/R-induced BMECs. Results: In vivo experiments demonstrated that THSWD effectively mitigated cerebral damage and restored neurological function in MCAO/R rats. THSWD significantly enhanced CD31, Ang1, PDGFB, and PDGFR-β expression levels, likely associated with improved glucose, pyruvate, and ATP levels, along with reduced lactate and lactate/pyruvate ratios. In vitro findings suggested that THSWD may boost the expression of mature angiogenesis factors (VEGFA, Ang1, and PDGFB) by activating glycolysis, increasing glucose uptake and augmenting lactate, pyruvate, and ATP content, thus accelerating mature angiogenesis. Conclusion: THSWD could alleviate CIRI by activating the glycolysis pathway to promote mature angiogenesis. Targeting the glycolysis-mediated mature angiogenesis alongside THSWD therapy holds promise for IS treatment.

Notoginsenoside R1 decreases intraplaque neovascularization by governing pericyte-endothelial cell communication via Ang1/Tie2 axis in atherosclerosis.

Atherosclerosis represents the major cause of mortality worldwide and triggers higher risk of acute cardiovascular events. Pericytes-endothelial cells (ECs) communication is orchestrated by ligand-receptor interaction generating a microenvironment which results in intraplaque neovascularization, that is closely associated with atherosclerotic plaque instability. Notoginsenoside R1 (R1) exhibits anti-atherosclerotic bioactivity, but its effect on angiogenesis in atherosclerotic plaque remains elusive. The aim of our study is to explore the therapeutic effect of R1 on vulnerable plaque and investigate its potential mechanism against intraplaque neovascularization. The impacts of R1 on plaque stability and intraplaque neovascularization were assessed in ApoE-/- mice induced by high-fat diet. Pericytes-ECs direct or non-direct contact co-cultured with VEGF-A stimulation were used as the in vitro angiogenesis models. Overexpressing Ang1 in pericytes was performed to investigate the underlying mechanism. In vivo experiments, R1 treatment reversed atherosclerotic plaque vulnerability and decreased the presence of neovessels in ApoE-/- mice. Additionally, R1 reduced the expression of Ang1 in pericytes. In vitro experiments demonstrated that R1 suppressed pro-angiogenic behavior of ECs induced by pericytes cultured with VEGF-A. Mechanistic studies revealed that the anti-angiogenic effect of R1 was dependent on the inhibition of Ang1 and Tie2 expression, as the effects were partially reversed after Ang1 overexpressing in pericytes. Our study demonstrated that R1 treatment inhibited intraplaque neovascularization by governing pericyte-EC association via suppressing Ang1-Tie2/PI3K-AKT paracrine signaling pathway. R1 represents a novel therapeutic strategy for atherosclerotic vulnerable plaques in clinical application.

Topical Clonidine Accelerates Cutaneous Wound Healing in Diabetic Rats by Regulating the Expression of Related Cytokine Signaling.

Based on the analgesic and anti-inflammatory effects of clonidine in previous studies, we hypothesized that clonidine could accelerate wound healing in rats by regulating the expression of related cytokines. In this study, the wound healing effect of clonidine was evaluated using an excision wound model in diabetic rats and a HaCaT cell model. The wounds were treated daily with topical clonidine. The results analyzed by ImageJ2 software show that the wounds of the rats that were treated with 15 ng/mL clonidine recovered faster, and the wound size was also significantly reduced compared to the control group. Western blot assays determined that clonidine induced an increase in the expression of vascular growth factors, namely, Ang-1, Ang-2, and VEGF. Moreover, clonidine demonstrated a rescuing effect on JAK2 within the JAK/STAT pathway by inhibiting SOCS3 expression, leading to decreased SOCS3 levels and increased expression of JAK2 and phospho-STAT3. Histopathological analysis revealed that clonidine promoted complete epithelial repair and minimized inflammation in skin tissue. Additionally, clonidine stimulated HaCaT cell proliferation in vitro and enhanced cellular energy levels in the presence of AGEs. In conclusion, clonidine promoted vascular growth and wound healing by stimulating the expression of cytokines that are beneficial for wound healing.

Disturbance in cerebral blood microcirculation and hypoxic-ischemic microenvironment are associated with the development of brain metastasis.

Brain metastases (BM) constitute an increasing challenge in oncology due to their impact on neurological function, limited treatment options, and poor prognosis. BM occur through extravasation of circulating tumor cells across the blood-brain barrier. However, the extravasation processes are still poorly understood. We here propose a brain colonization process which mimics infarction-like microenvironmental reactions, that is dependent on Angiopoietin (Ang-2) and vascular endothelial growth factor (VEGF). In this study, intracardiac BM models were used, and cerebral blood microcirculation was monitored by 2-photon microscopy through a cranial window. BM formation was observed using cranial magnetic resonance, bioluminescent imaging, and post-mortem autopsy. Ang-2/VEGF targeting strategies and Ang-2 gain-of-function (GOF) mice were employed to interfere with BM formation. In addition, vascular and stromal factors as well as clinical outcome were analyzed in BM patients. Blood vessel occlusions by cancer cells were detected, accompanied by significant disturbances of cerebral blood microcirculation, and focal stroke-like histological signs. Cerebral endothelial cells showed an elevated Ang-2 expression both in mouse and human BM. Ang-2 GOF resulted in an increased BM burden. Combined anti-Ang-2/anti-VEGF therapy led to a decrease in brain metastasis size and number. Ang-2 expression in tumor vessels of established human brain metastases negatively correlated with survival. Our observations revealed a relationship between disturbance of cerebral blood microcirculation and brain metastasis formation. This suggests that vessel occlusion by tumor cells facilitates brain metastatic extravasation and seeding, while combined inhibition of microenvironmental effects of Ang-2 and VEGF prevent the outgrowth of macrometastases.

NRASQ61R mutation drives elevated angiopoietin-2 expression in human endothelial cells and a genetic mouse model.

Angiopoietin-2 (Ang-2) is increased in the blood of patients with kaposiform lymphangiomatosis (KLA) and kaposiform hemangioendothelioma (KHE). While the genetic causes of KHE are not clear, a somatic activating NRASQ61R mutation has been found in the lesions of KLA patients. Our study tested the hypothesis that the NRASQ61R mutation drives elevated Ang-2 expression in endothelial cells. Ang-2 was measured in human endothelial progenitor cells (EPC) expressing NRASQ61R and a genetic mouse model with endothelial targeted NRASQ61R. To determine the signaling pathways driving Ang-2, NRASQ61R EPC were treated with signaling pathway inhibitors. Ang-2 levels were increased in EPC expressing NRASQ61R compared to NRASWT by Western blot analysis of cell lysates and ELISA of the cell culture media. Ang-2 levels were elevated in the blood of NRASQ61R mutant mice. NRASQ61R mutant mice also had reduced platelet counts and splenomegaly with hypervascular lesions, like some KLA patients. mTOR inhibitor rapamycin attenuated Ang-2 expression by NRASQ61R EPC. However, MEK1/2 inhibitor trametinib was more effective blocking increases in Ang-2. Our studies show that the NRASQ61R mutation in endothelial cells induces Ang-2 expression in vitro and in vivo. In cultured human endothelial cells, NRASQ61R drives elevated Ang-2 through MAP kinase and mTOR-dependent signaling pathways.

Effects of 4-Hydroxybenzaldehydeon on Interactions With Neurovascular Unit-Related Cells

4-hydroxybenzaldehyde (4-HBd) is one of the active compounds with neuroprotective effects, which has been confirmed to have anti cerebral ischemical reperfusion injury (CIRI) effect in previous study. In this study, we explored the protective effect of 4-HBd on the neurovascular unit (NVU) after CIRI and its mechanism through in vivo and in vitro experiments. Establish rat of middle cerebral artery occlusion/reproduction (MCAO/R) model, transwell chamber was used to establish an in vitro primary cell co-culture model to simulate NVU, causing oxygen glucose deprivation/reperfusion (OGD/R) injury, simulating the pathological of CIRI in vivo. Longa 5-point method was used to evaluate the neurological function of the rats, and transmission electron microscope was used to observe the ultrastructural changes of NVU. Western Blot was used to detect the expression of neuronal protein in rat brain. The mRNA expressions of ang-1/tie-2 signaling pathway and bdnf/trkb signaling pathway were detected by qPCR. In vivo results showed that 4-HBd reduced neurological function scores and improved the ultrastructure of NVU after MCAO/R rats. 4-HBd could up-regulate the expression of microtubule associated protein-2 (Map-2), glial fibrillary acidic protein (GFAP) and occludin. In vitro results showed that 4-HBd could activate ang-1/tie-2 signaling pathway, increased occludin mRNA expression and protect the blood brain barrier (BBB). 4-HBd can activate bdnf/trkb signaling pathway, up-regulate map-2 mRNA expression, and promote neuronal repair. In vitro and in vivo results indicated 4-HBd can affect the ang-1/tie-2 and bdnf/trkb signaling pathways, and BBB damage is alleviated and NVU homeostasis is maintained to improve CIRI.

Kidney Mesenchymal Stem Cell Differentiation: Effect of Scaffold and Basic Fibroblast Growth Factor.

Background: Chronic kidney disease (CKD) poses a global health challenge, and it needs alternative therapeutic approaches for patients with end-stage renal disease (ESRD). Although organ transplantation is effective, it faces challenges such as declining quality of life, immunological responses, transplant rejection, and donor shortages. Tissue engineering, by using suitable scaffolds, cells, and growth factors, emerges as a promising treatment option for kidney regeneration. Experiment: We precisely decellularized scaffold, derived from rat kidneys while maintaining its native three-dimensional (3D) architecture. The efficiency of decellularization was evaluated through histological examinations, including hematoxylin and eosin, periodic acid-Schiff, and DAPI staining, as well as scanning electron microscopy. The scaffolds were then recellularized with kidney mesenchymal stem cells (kMSCs), and their adhesion, proliferation, and differentiation were assessed over 1, 2, and 3 weeks. The expression of specific renal markers, including Wt-1, ZO-1, AQP-1, and ANG-1, was examined through quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in monolayer and 3D cultures. Results: The infiltration rate of cells into the scaffold increased in a time-dependent manner, and the expression of specific renal markers significantly increased, demonstrating successful differentiation of kMSCs within the scaffold. The application of basic fibroblast growth factor (bFGF) could intensify the expression of kidney-specific genes. Conclusions: The study highlighted the importance of preserving the 3D architecture of the scaffold during decellularization to achieve optimal cellular responses. Moreover, the capacity of mesenchymal stem cells in recellularized scaffolds facilitated tissue regeneration.

Improved therapy for clear cell renal cell carcinoma: beta-hydroxybutyrate and quercetin target hypoxia-induced angiogenesis and multidrug resistance.

Clear cell renal cell carcinoma (ccRCC) is a form of kidney cancer characterized by dysregulated angiogenesis and multidrug resistance. Hypoxia-induced tumor progression plays a crucial role in ccRCC pathogenesis. Beta-hydroxybutyrate (BHB) and quercetin (QCT) have shown potential in targeting angiogenesis and drug resistance in various cancer types. This study investigates the combined effects of BHB and QCT in hypoxia-induced Caki-1 cells. Caki-1 cells were subjected to normoxic and hypoxic conditions and treated with BHB, QCT, or a combination of both. Cell-viability was assessed using the MTT assay, and mRNA expression levels of key angiogenesis-related genes (HIF-1α/2α, VEGF, Ang-1, Ang-2, and MDR4) were quantified through real-time PCR during 24 and 48h. BHB and QCT treatments, either alone or in combination, significantly reduced cell-viability in Caki-1 cells (p < 0.05). Moreover, the combined therapy demonstrated a potential effect in downregulating the expression of angiogenesis-related genes and MDR4 in hypoxia-induced cells, with a marked reduction in HIF-1α/2α, VEGF, Ang-1, and MDR4 expression (p < 0.05). The expression of Ang-2 increases significantly in presence of BHB combined QCT treatment. This study highlights the promising potential of a combination therapy involving BHB and QCT in mitigating angiogenesis and MDR4 expression in hypoxia-induced ccRCC cells. These findings support further investigation into the underlying mechanisms and warrant clinical studies to evaluate the therapeutic value of this combined treatment for ccRCC patients. This research provides new insights into addressing the challenges posed by angiogenesis and drug resistance in ccRCC.

Hypoxic bone marrow mesenchymal stem cell exosomes promote angiogenesis and enhance endometrial injury repair through the miR-424-5p-mediated DLL4/Notch signaling pathway.

Currently, bone marrow mesenchymal stem cells (BMSCs) have been reported to promote endometrial regeneration in rat models of mechanically injury-induced uterine adhesions (IUAs), but the therapeutic effects and mechanisms of hypoxic BMSC-derived exosomes on IUAs have not been elucidated. To investigate the potential mechanism by which the BMSCS-derived exosomal miR-424-5p regulates IUA angiogenesis through the DLL4/Notch signaling pathway under hypoxic conditions and promotes endometrial injury repair. The morphology of the exosomes was observed via transmission electron microscopy, and the expression of exosome markers (CD9, CD63, CD81, and HSP70) was detected via flow cytometry and Western blotting. The expression of angiogenesis-related genes (Ang1, Flk1, Vash1, and TSP1) was detected via RT‒qPCR, and the expression of DLL4/Notch signaling pathway-related proteins (DLL4, Notch1, and Notch2) was detected via Western blotting. Cell proliferation was detected by a CCK-8 assay, and angiogenesis was assessed via an angiogenesis assay. The expression of CD3 was detected by immunofluorescence. The endometrial lesions of IUA rats were observed via HE staining, and the expression of CD3 and VEGFA was detected via immunohistochemistry. Compared with those in exosomes from normoxic conditions, miR-424-5p was more highly expressed in the exosomes from hypoxic BMSCs. Compared with those in normoxic BMSC-derived exosomes, the proliferation and angiogenesis of HUVECs were significantly enhanced after treatment with hypoxic BMSC-derived exosomes, and these effects were weakened after inhibition of miR-424-5p. miR-424-5p can target and negatively regulate the expression of DLL4, promote the expression of the proangiogenic genes Ang1 and Flk1, and inhibit the expression of the antiangiogenic genes Vash1 and TSP1. The effect of miR-424-5p can be reversed by overexpression of DLL4. In IUA rats, treatment with hypoxic BMSC exosomes and the miR-424-5p mimic promoted angiogenesis and improved endometrial damage. The hypoxic BMSC-derived exosomal miR-424-5p promoted angiogenesis and improved endometrial injury repair by regulating the DLL4/Notch signaling pathway, which provides a new idea for the treatment of IUAs.

Quemeiteng granule relieves goiter by suppressing thyroid microvascular endothelial cell proliferation and angiogenesis via miR-217-5p-mediated targeting of FGF2-induced regulation of the ERK pathway

Ethnopharmacological relevanceGoiters are enlargements of the thyroid gland and are a global public issue. Quemeiteng granule (QMTG) is a traditional Chinese medicine (TCM) formula used to treat goiter in Yunnan Province. However, the effectiveness and underlying mechanism of these treatments have not been fully elucidated. Aim of the studyThis study aimed to investigate the therapeutic effects of QMTG on goiter and the downstream regulatory mechanisms. Materials and methodsIn this study, we first evaluated the antigoiter efficacy of QMTG through biochemical indices [body weight, thyroid coefficient, triiodothyronine (T3), thyroxine (T4), free triiodothyronine (FT3), free thyroxine (FT4), and thyroid stimulating hormone (TSH)] and hematoxylin-eosin (HE) staining in a Propylthiouracil (PTU)-induced model. Based on microRNA sequencing (miRNA-seq) and bioinformatics analysis, key miRNA was screened out. A dual-luciferase reporter assay was performed to confirm the transcriptional regulation of the target gene by the miRNA. The viability of rat thyroid microvascular endothelial cells (RTMECs) and human thyroid microvascular endothelial cells (HTMECs) was assessed using the CCK-8 assays. The migration and angiogenesis of RTMECs and HTMECs were visualized through tube formation and wound scratch assays. Proteins involved in angiogenesis and the ERK pathway were assessed via Western blotting. ResultsQMTG significantly increased body weight, decreased the thyroid coefficient, increased the levels of T3, T4, FT3 and FT4 and reduced TSH levels in rats with goiter. QMTG also promoted the morphological recovery of thyroid follicles. MiR-217-5p was identified as a key miRNA. Our studies revealed that miR-217-5p directly targets FGF2 and that QMTG promotes the recovery of thyroid hormone (TH) levels and morphological changes in the thyroid, suppresses thyroid microvascular endothelial cell vitality, tube formation and migration, and reduces the expression of VEGF, Ang-1 and VCAM-1 triggered by miR-217-5p, thereby inhibiting the Ras/MEK/ERK cascade through FGF2. ConclusionsOur experiments demonstrated that the QMTG had therapeutic effects on goiter. These effects were attributed to the inhibition of ERK pathway-induced proliferation and angiogenesis through the targeting of FGF2 by miR-217-5p.

Experimental study of tetramethylpyrazine-loaded electroconductive hydrogel on angiogenesis and neuroprotection after spinal cord injury

To explore the mechanisms for repairing spinal cord injury (SCI) with tetramethylpyrazine-loaded electroconductive hydrogel (hereinafter referred to as "TGTP"). A total of 72 female Sprague-Dawley rats were randomly divided into 4 groups: sham operation group (group A), SCI group (group B), SCI+electroconductive hydrogel group (group C), and SCI+TGTP group (group D). Only the vertebral plate was removed in group A, while the remaining groups were subjected to a whole transection model of spinal cord with a 2 mm gap in the lesions. The recovery of hindlimb motor function was evaluated by Basso, Beattie, Bresnahan (BBB) score and modified Rivlin-Tator inclined plate test before operation and at 1, 3, 7, 14, and 28 days after operation, respectively. Animals were sacrificed at 7 days and 28 days after modeling. Neovascularisation was observed by immunofluorescence staining of CD31 and the expression levels of angiopoietin 1 (Ang-1) and Tie-2 were assessed by Western blot assay. At 28 days postoperatively, the expression levels of pro-angiogenic related proteins, including platelet-derived growth factor B (PDGF-B), PDGF receptor β (PDGFR-β), vascular endothelial growth factor A (VEGF-A), and VEGF receptor 2 (VEGFR-2), were also assessed by Western blot. The fibrous scar in the injured area was assessed using Masson staining, while neuronal survival was observed through Nissl staining. Furthermore, LFB staining was utilized to detect myelin distribution and regeneration. Immunofluorescence and Western blot assay were employed to evaluate the expression of neurofilament 200 (NF200). The hindlimb motor function of rats in each group gradually recovered from the 3rd day after operation. The BBB score and climbing angle in group D were significantly higher than those in group B from 3 to 28 days after operation, and significantly higher than those in group C at 14 days and 28 days after operation ( P<0.05). Masson staining showed that the collagen volume fraction in groups B-D were significantly higher than that in group A, and that in group D was significantly lower than that in groups B and C ( P<0.05); a small amount of black conductive particles were scattered at the broken end in group D, and the surrounding collagen fibers were less than those in group C. Nissl and LFB staining showed that the structure of neurons and myelin sheath in the injured area of spinal cord in group D was relatively complete and continuous, and the number of Nissl bodies and the positive area of myelin sheath in group D were significantly better than those in groups B and C ( P<0.05). NF200 immunofluorescence staining and Western blot assay results showed that the relative expression of NF200 protein in group D was significantly higher than that in groups B and C ( P<0.05). CD31 immunofluorescence staining showed that the fluorescence intensity of group D was better than that of groups B and C at 28 days after operation, and tubular or linear neovascularization could be seen. The relative expressions of Ang-1 and Tie-2 proteins in group D were significantly higher than those in groups B and C at 7 and 28 days after operation ( P<0.05). The relative expressions of PDGF-B and PDGFR-β proteins in group D were significantly higher than those in groups B and C, and group B was significantly higher than group C at 28 days after operation ( P<0.05). The relative expressions of VEGF-A and VEGFR2 proteins in group D were higher than those in groups B and C, showing significant difference when compared with group B ( P<0.05), but only the expression of VEGF-A protein was significantly higher than that in group C ( P<0.05). There was significant difference only in VEGFR-2 protein between groups B and C ( P<0.05). TGTP may enhance the revascularization of the injured area and protect the neurons, thus alleviating the injury of spinal cord tissue structure and promoting the recovery of neurological function after SCI in rats.

MiR-221-5p regulates blood–brain barrier dysfunction through the angiopoietin-1/-2/Tie-2 signaling axis after subarachnoid hemorrhage

ABSTRACT Aim To explore the potential role of microRNA miR-221-5p on the angiopoietin-1 (Ang-1)/Ang-2/Tie-2 signaling axis after subarachnoid hemorrhage (SAH) in a rat model. Methods Aspects of the rat’s behavior were measured using the Kaoutzanis scoring system to test neurological responses. This included feeding behavior, body contraction, motor, and eye-opening responses. Brain sections were studied using transmission electron microscopy and Evans blue extravasation. Levels of Ang-1, Ang-2, and Tie-2 were determined by Western blot, while miR-221-5p was quantified using stem-loop real-time quantitative PCR (RT-qPCR). Results The SAH group responded worse to the neurological response test than the sham-operated group. The intercellular space was widened in the SAH group, but not in the sham-operated group. Evans blue dye leaked significantly more into brain tissue cells of the SAH group. Stem-loop qRT-PCR showed elevated miR-221-5p levels. Additionally, Ang-1 and Tie-2 were reduced but Ang-2 expression was increased after SAH. This led to a significant reduction of the Ang-1/Ang-2 ratio in the brain tissue, which was associated with the destruction of the blood-brain barrier. Conclusion The data indicate that miR-221-5p might regulate blood-brain barrier dysfunction through the Ang-1/Ang-2/Tie-2 signaling axis, suggesting that it should be further investigated as a potential novel biomarker.